Heme Oxygenase-1 Deficiency and Oxidative Stress

International Journal of Molecular Sciences

Review Heme Oxygenase-1 Deﬁciency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models

Akihiro Yachie

Division of Medical Safety, Kanazawa University Hospital, Kanazawa 920-8641, Japan; [email protected]; Tel.: +81-76-265-2073

Abstract: Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases. Keywords: heme oxygenase; HO-1 deficiency; oxidative stress; inflammation Citation: Yachie, A. Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and 1. Background Animal Models. Int. J. Mol. Sci. 2021, 22, 1514. https://doi.org/ Heme is a major component of hemoglobin (Hb), and is a product of erythrocyte 10.3390/ijms22041514 destruction. Heme is constantly produced in vivo through the destruction of both senescent erythrocytes under physiological conditions, and abnormal erythrocytes under pathological Academic Editor: József Balla conditions. Heme is extremely toxic to cells, so constitutive mechanisms exist to cancel the Received: 17 January 2021 toxic effects of heme [1,2]. Serum haptoglobin (Hp) binds to free Hb efficiently [3,4] and Accepted: 28 January 2021 the resulting Hb-Hp complex is promptly taken up by phagocytes and hepatocytes, which Published: 3 February 2021 express a receptor for the complex that is currently known as CD163 [5–7]. Heme oxygenase (HO)-1 constitutes one of the three isozymes of HO, and catalyzes the degradation of heme Publisher’s Note: MDPI stays neutral into biliverdin, carbon monoxide (CO), and free iron, each of which somehow exert potent with regard to jurisdictional claims in anti-oxidative stress and anti-inflammatory functions [8,9]. For this reason, HO-1 exerts published maps and institutional affil- protective properties in a broad range of human diseases, including neurodegenerative iations. diseases, cardiovascular diseases, cancer, metabolic diseases, iron metabolism disorders and various inflammatory diseases [10] (Figure1). Among the heme degradation products, CO plays a central role and acts on cellular metabolisms to protect cells from oxidative stress and regulate the production of inflam- Copyright: © 2021 by the author. matory molecules [11–13]. CO directly controls the inflammatory state of a given tissue, Licensee MDPI, Basel, Switzerland. and at the same time regulates the microcirculation within target organs by acting as a This article is an open access article gaseous mediator [14,15]. Among the three isozymes, HO-1 is the only protein rapidly distributed under the terms and induced upon stimulation with various oxidative stresses [14,16]. Therefore, any defect in conditions of the Creative Commons the function of HO-1 would be expected to lead to uncontrollable inflammation in response Attribution (CC BY) license (https:// to certain exogenous insults, such as infection and hemolysis. creativecommons.org/licenses/by/ 4.0/).

Int. J. Mol. Sci. 2021, 22, 1514. https://doi.org/10.3390/ijms22041514 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17

After Yachie et al. reported the first human case of HO-1 deficiency [17,18], 5 cases with identical mutation were recognized in India [19–21]. Additional cases with nonsense mutations and a case with a missense mutation have recently been reported [22–24]. In Int. J. Mol. Sci. 2021,this22, 1514 short review, I describe the clinical characteristics and laboratory features of these 2 of 16 cases and propose denominators for this extremely rare genetic disease.

Figure 1. HemeFigure oxygenase 1. Heme oxygenase (HO)-1 and (HO)-1 the hemeand the degradation heme degradation pathway. pathway. Free hemoglobin Free hemoglobin (Hb) quickly(Hb) binds with haptoglobinquickly (Hp) to binds form awith molecular haptoglobin complex (Hp) (Hb-Hp). to form a Hb-Hp molecular is rapidly complex incorporated (Hb-Hp). Hb-Hp through is therapidly scavenger receptor, now knownincorporated as CD163, expressed through onthe the scavenger cell surface receptor, of tissue now macrophages known as CD163, such expre as hepaticssed on Kupffer the cell cells. surface Heme derived from Hb is degradedof tissue macrophages by HO-1 into such free ironas hepatic (Fe++), Kupffer carbon monoxidecells. Heme (CO) derived and from biliverdin. Hb is Biliverdindegraded by is furtherHO-1 degraded ++ into bilirubininto by free biliverdin iron (Fe reductase.), carbon All monoxide these heme (CO) degradation and biliverdin. products Biliverdin are known is further to exert degraded potent into anti-inflammatory bili- and anti-oxidativerubin by stress biliverdin functions, reductase. which inAll turn these play heme significant degradation roles inproducts preventing are known various to chronic exert potent diseases. anti-inflammatory and anti-oxidative stress functions, which in turn play significant roles in preventing various chronicAfter diseases. Yachie et al. reported the first human case of HO-1 deficiency [17,18], 5 cases with identical mutation were recognized in India [19–21]. Additional cases with nonsense 2. Case Reportsmutations of Human and HO-1 a case Deficiency with a missense mutation have recently been reported [22–24]. In this Althoughshort numerous review, studies I describe have the been clinical published characteristics utilizing and animal laboratory models features and in of these cases vitro experimentsand with propose regard denominators to the role of for HO-1 this in extremely vivo, only rare a limited genetic number disease. of human cases have been reported to date. Although the number of such cases remains limited, clinical descriptions2. Case of Reports these human of Human cases HO-1 of HO-1 Deficiency deficiency highlight the critical roles played by this enzymeAlthough in protecting numerous cells studies and organs have been from published oxidative utilizingstress and animal inflamma- models and in vitro tion. experiments with regard to the role of HO-1 in vivo, only a limited number of human cases have been reported to date. Although the number of such cases remains limited, clinical 2.1. The First Casedescriptions of these human cases of HO-1 deficiency highlight the critical roles played by In 1999, Yachiethis enzyme et al. reported in protecting on a cells26-month-old and organs boy from who oxidative had been stress admitted and inflammation. with recurrent fever, generalized erythematous rash, and joint pain [17]. The combination of 2.1. The First Case fever, erythematous rash, and joint pain suggested that the patient suffered from a childhood chronic inflammatoryIn 1999, Yachie illness etsuch al. reportedas systemic on juvenile a 26-month-old idiopathic boy arthritis who had (sJIA) been or admitted with chronic infantilerecurrent neurological fever, cutaneous generalized and erythematous articular syndrome rash, and (alternatively joint pain called [17]. Thene- combination onatal-onset multisystemof fever, erythematous inflammatory rash, disease). and joint The pain latter suggested condition that is now the patientknown sufferedas from a cryopyrin-associatedchildhood periodic chronic syndrome. inflammatory However, illness neither such as of systemic these conditions juvenile idiopathic could ex- arthritis (sJIA) plain the uniqueor chronicclinical infantilefeatures neurologicaland extraordinary cutaneous laboratory and articular findings syndrome seen in this (alternatively pa- called tient. The patientneonatal-onset had characteristic multisystem facial feat inflammatoryures such as disease).a flat nasal The bridge, latter frontal condition boss- is now known as cryopyrin-associated periodic syndrome. However, neither of these conditions could ing, and prominent edema of the eyelids. Although the liver was markedly enlarged, the explain the unique clinical features and extraordinary laboratory findings seen in this spleen was absent. Unlike most patients with asplenia, this patient did not have any form patient. The patient had characteristic facial features such as a flat nasal bridge, frontal bossing, and prominent edema of the eyelids. Although the liver was markedly enlarged, the spleen was absent. Unlike most patients with asplenia, this patient did not have any form of congenital heart disease. His brother and sister appeared healthy, but the mother had experienced two intrauterine fetal deaths. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 17

of congenital heart disease. His brother and sister appeared healthy, but the mother had Int. J. Mol. Sci. 2021, 22, 1514 experienced two intrauterine fetal deaths. 3 of 16 The most significant characteristics of his laboratory data were prominent increases in white blood cell count (51,600/μL) and platelet count (226 × 104/μL). This marked leukocytosis and thrombocytosis persisted throughout the course of the illness. The patient The most significant characteristics of his laboratory data were prominent increases 6 showedin white significant blood cell anemia count (51,600/ with anµ L)erythrocyte and platelet count count of 1.48 (226 × 1010/4μ/Lµ L).and This a hemoglobin marked concentrationleukocytosis and of 4.9 thrombocytosis g/dL. Peripheral persisted blood throughout smear showed the course numerous of the illness. fragmented The patient erythrocytes,showed Howell-Jolly significant anemia bodies with and an nucleated erythrocyte red count blood of cells. 1.48 ×Serum106/µ ironL and concentration a hemoglobin was normalconcentration at 64 μ ofg/dL. 4.9 g/dL. The level Peripheral of lactate blood dehydrogenase smear showed numerous(LDH) was fragmented extremely erythro- elevated (17,470cytes, Howell-JollyIU/L, normal bodies range; and 196–355) nucleated and red aspartate blood cells. aminotransferase Serum iron concentration (AST) was also was in- creasednormal (442 at 64 IU/L,µg/dL. normal The level range; of lactate 9–42),dehydrogenase but alanine aminotransferase (LDH) was extremely (ALT) elevated remained within(17,470 normal IU/L, normallimits. Serum range; 196–355)ferritin was and 780 aspartate ng/mL aminotransferase (normal range; 26–280). (AST) was Marked also in- ab- normalitiescreased (442 were IU/L, noted normal in parameters range; 9–42), of butboth alanine the coagulation aminotransferase and fibrinolysis (ALT) remained systems, althoughwithin normal he did limits.not show Serum any ferritinapparent was bleedi 780 ng/mLng tendency (normal or signs range; of 26–280). accelerated Marked coagulation.abnormalities Fibrinogen were was noted 109 in mg/dL parameters (normal of both range; the coagulation196–356), fibrin and fibrinolysis degradation systems, product wasalthough 300.1 heμg/mL did not (normal show any range; apparent <5), bleedingd-dimer tendencywas 186.1 or signsμg/mL of accelerated(normal range; coagula- <2.5), thrombin-antithrombintion. Fibrinogen was 109 complex mg/dL (normalwas 202.2 range; μg/L 196–356), (normal fibrin range; degradation <3), and plasmin-2 product was plas- 300.1 µg/mL (normal range; <5), d-dimer was 186.1 µg/mL (normal range; <2.5), thrombin- min inhibitor complex was 22.3 μg/mL (normal range; <0.8). Thrombomodulin was 12 antithrombin complex was 202.2 µg/L (normal range; <3), and plasmin-2 plasmin inhibitor ng/mL (normal range; <3.5) and von Willbrand factor was 580% (normal range; 32–115). complex was 22.3 µg/mL (normal range; <0.8). Thrombomodulin was 12 ng/mL (normal Hyperlipidemiarange; <3.5) and was von another Willbrand prominent factor was finding, 580% (normal with triglycerides range; 32–115). at 638 Hyperlipidemia mg/dL (normal range;was another 32–115) prominent and total cholesterol finding, with at triglycerides552 mg/dL (normal at 638 mg/dL range; (normal128–219), range; showing 32–115) a pre- dominanceand total cholesterol of low-density at 552 lipoprotein mg/dL (normal cholesterol. range; 128–219), showing a predominance of low-densitySerum specimens lipoprotein looked cholesterol. quite peculiar, appearing turbid with a brownish tint (Fig- ure 2A).Serum This specimens was not due looked to mechanical quite peculiar, damage appearing to the erythrocytes turbid with athrough brownish any tint trau- matic(Figure procedures,2A) . This wasor the not result due toof mechanicalex vivo destruction damage to of the fragile erythrocytes erythrocytes. through Repeated any bloodtraumatic sampling procedures, yielded or similar the result results. of ex Venous vivo destruction blood samples of fragile were erythrocytes. spun down Repeated right after venipunctureblood sampling and yielded serum similaralways results.appeared Venous the same. blood Given samples the dark were coloration spun down of right the patient’safter venipunctureserum samples, and certain serum alwayshemoglobinopat appearedhies the same. were Givensuspected. the dark Hemolysate coloration of of the erythrocytesthe patient’s was serum transparent samples, certain and showed hemoglobinopathies only two distinct were peaks suspected. corresponding Hemolysate to ofoxy- hemoglobinthe erythrocytes (OxyHb), was transparentat 541 nm and and 576 showed nm (F onlyigure two 2B). distinct In addition peaks corresponding to these two peaks to oxyhemoglobin (OxyHb), at 541 nm and 576 nm (Figure2B). In addition to these two peaks for OxyHb, serum from the patient showed a third unique peak at 631 nm, corresponding for OxyHb, serum from the patient showed a third unique peak at 631 nm, corresponding to methemoglobin (MetHb). The gross appearance of the serum combined with the results to methemoglobin (MetHb). The gross appearance of the serum combined with the results of absorption spectrum analysis suggested that heme in the form of OxyHb and MetHb of absorption spectrum analysis suggested that heme in the form of OxyHb and MetHb waswas markedly markedly increased increased in thethe patient’spatient’s serum serum (Figure (Figure2C). 2C).

FigureFigure 2. 2. SerumSerum characteristics characteristics of of HO-1 HO-1 deficiency. deﬁciency. Patient Patient serum serum separated separated immediately immediately after after bloodblood drawing drawing is is characterized characterized byby aa turbid,turbid, dark-brown dark-brown color color (A ().A Fresh). Fresh hemolysate hemolysate of the of the patient’s patient’serythrocytes erythrocytes shows shows only theonly peaks the peaks of oxyhemoglobin of oxyhemoglobin (OxyHb) (OxyHb) (B). Absorption (B). Absorption spectrum spectrum of the of patient’s serum reveals inclusion of large amounts of both OxyHb and methemoglobin (MetHb) (C).

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the patient’s serum reveals inclusion of large amounts of both OxyHb and methemoglobin Int. J. Mol. Sci. 2021, 22, 1514 (MetHb) (C). 4 of 16

These results indicated that either massive hemolysis was constantly taking place in vivo or that Hb was accumulating in serum due to defects in the Hb catabolic pathway. These results indicated that either massive hemolysis was constantly taking place in vivo Further study revealed that serum Hp concentration was extremely elevated (800–1200 or that Hb was accumulating in serum due to defects in the Hb catabolic pathway. Fur- mg/dL, normal range; 40–180 mg/dL) and a large amount of Hb-Hp complex was detected ther study revealed that serum Hp concentration was extremely elevated (800–1200 mg/dL, in a urine sample from the patient. These data suggested that the Hb-Hp complex was normal range; 40–180 mg/dL) and a large amount of Hb-Hp complex was detected in a somehow bypassing the normal scavenger system and overflowing into urine. Repeated urine sample from the patient. These data suggested that the Hb-Hp complex was somehow measurements of serum bilirubin concentration were always low, at 0.1–0.3 mg/dL. bypassing the normal scavenger system and overflowing into urine. Repeated measurements Hemopexin was undetectable by immunoelectrophoresis, indicating the presence of ac- of serum bilirubin concentration were always low, at 0.1–0.3 mg/dL. Hemopexin was un- tive hemolysis in vivo. Serum heme concentration was extremely high, at 490 μM (normal detectable by immunoelectrophoresis, indicating the presence of active hemolysis in vivo. range; <1 μM). Results for both direct and indirect Coombs’ tests were negative on re- Serum heme concentration was extremely high, at 490 µM (normal range; <1 µM). Results for peated occasions. All these data together were highly suggestive of abnormalities in either both direct and indirect Coombs’ tests were negative on repeated occasions. All these data thetogether process were of hepatic highly suggestiveuptake of Hb-Hp of abnormalities complex or in eitherthe heme the degradation process of hepatic pathway uptake (Fig- of ureHb-Hp 3). complex or the heme degradation pathway (Figure3).

FigureFigure 3. 3. ProposedProposed mechanism mechanism of of MetHb MetHb accumulation accumulation in in blood. blood. Under Under normal normal conditions, conditions, small small amountsamounts of of Hb Hb derived derived from from hemolysis hemolysis are arerapidly rapidly bound bound by circulating by circulating Hp, forming Hp, forming Hb-Hp Hb-Hp complexes,complexes, which which are are rapidly rapidly take takenn up up by by the the liver liver through through CD163 CD163 (A ().A When). When massive massive hemolysis hemolysis occurs,occurs, as as in in the the case case of of hemolytic hemolytic anemia, anemia, larg largee amounts amounts of offree free Hb Hb are are bound bound by byserum serum Hp Hp and and transferredtransferred to to the the liver liver ( (BB).). This This results results in in a a significant significant reduction ofof serumserum HpHp andand increasedincreased indirectindi- rectbilirubin bilirubin (Bil) (Bil) derived derived from from the hemethe heme degradation degradation pathway. pathway. In HO-1-deficient In HO-1-deficient patients, patients, both both OxyHb OxyHb and MetHb bound to Hp accumulate within the serum. Despite the apparent intravascular and MetHb bound to Hp accumulate within the serum. Despite the apparent intravascular hemolysis, hemolysis, total Hp content is significantly increased, serum bilirubin level remains low, and over- total Hp content is significantly increased, serum bilirubin level remains low, and overflow of Hb-Hp flow of Hb-Hp complex into the urine is seen (C). complex into the urine is seen (C). Immediately before we reported this case of human HO-1 deficiency, Poss and Immediately before we reported this case of human HO-1 deficiency, Poss and Tone- Tonegawa had made monumental reports describing the characteristics of HO-1-deficient gawa had made monumental reports describing the characteristics of HO-1-deficient mice [25,26]. Those results shared many findings in common with our patient, including mice [25,26]. Those results shared many findings in common with our patient, including se- severe anemia due to defective iron reuse, tissue iron deposition, enhanced cell injury due vere anemia due to defective iron reuse, tissue iron deposition, enhanced cell injury due to tooxidative oxidative stress stress and and accelerated accelerated tissue tissue inflammation. inflammation. Immunohistochemical Immunohistochemical analysis analysis of theof theliver liver biopsy biopsy specimen specimen showed showed that that Kupffer Kupffer cells cells did did not producenot produce HO-1 HO-1 in the in patient’sthe patient’s liver. liver.Exposure Exposure of the of patient’s the patient’s monocytes monocytes to heavy to heavy metals, metals, such as such cadmium as cadmium or sodium or sodium arsenite, arsenite,did not inducedid not HO-1 induce protein. HO-1 Immunoblottingprotein. Immunoblotting analysis revealed analysis thatrevealed lymphoblastoid that lympho- cell blastoidlines derived cell lines from derived the patient from did the not patient produce did HO-1not produce protein inHO-1 response protein to thesein response oxidative to thesestressors. oxidativeHMOX1 stressors.analysis HMOX1 revealed analysis that revealed the patient that was the apatient compound was a heterozygote compound het- for erozygotetwo HMOX1 for twomutations: HMOX1 the mutations: maternal allelethe maternal lacked theallele 2nd lacked exon, the while 2nd the exon, paternal while allele the paternalshowed allele a 2-base showed pair deletion a 2-base withinpair deletion the 3rd within exon. the 3rd exon. Pathological examination of the first case of HO-1 deficiency revealed characteristic tissue injury [18]. Notably, cellular injury was confined to selected organs and cell types, including the kidneys, liver, circulating monocytes, and vascular endothelial cells. In the kidneys, mild mesangial proliferation and thickening of the capillary loop were observed Int. J. Mol. Sci. 2021, 22, 1514 5 of 16

within glomeruli. Electron microscopy revealed marked swelling of the endothelial cells and detachment throughout the glomerular capillaries. In addition to glomerular damage, tubulointerstitial injury with tubular atrophy was signiﬁcant. The liver was massively enlarged and signiﬁcant amyloid accumulations had resulted in marked atrophy of hepatocytes. Scattered foci of iron deposits were observed in both the kidneys and liver. The majority of iron deposits were detected within renal tubular epithelial cells and hepatic parenchymal cells. The cytoplasm of circulating monocytes was vacuolated, and expres- sions of many monocyte surface antigens were markedly reduced. Although we could not see whether circulating monocytes from the patient expressed Hb-Hp receptor CD163, no expression was detectable on Kupffer cells from the liver of the patient [27].

2.2. The Indian Cases: 5 Independent Cases with an Identical HMOX1 Mutation After we reported the first case of human HO-1 deficiency, 5 cases from India were confirmed by HO-1 gene analysis (Radhakrishnan 2011 [19], Radhakrishnan 2011 [20], Gupta 2016 [21], and personal communications). Fever, absence of the spleen, hemolytic anemia, hematuria/proteinuria, and the absence of jaundice seem to be the common findings in those cases. All cases from India (Cases 2–6) displayed an identical homozygous mutation (p.R44X), indicating a founder effect of this particular mutation in that country. Although no functional study has been performed to confirm the impact of this particular mutation, it is a nonsense mutation and no HO-1 protein was detectable within the tissues examined. This mutation was thus suggested to be deleterious, resulting in impaired control of oxidative stress and inflammation. However, ages at disease onset and durations varied significantly between cases. Age at onset varied from 6 months to 15 years. Sur- prisingly, most patients remained asymptomatic for a long time until the onset of the first recognizable symptom. Case 2 (the first of the 5 Indian cases) remained healthy until she experienced prolonged fever at 15 years old. Growth and development were both normal and she had no significant past history. However, her condition deteriorated rapidly once the disease became apparent. Her terminal stage was complicated by hypertension and intracranial hemorrhage, as observed in the initial Japanese case. She died 5 months after the diagnosis, when fungal sepsis developed. Furthermore, two other reported cases, Case 3 and Case 6 also showed rapid deterioration and died soon after the onset of symptoms. The variability of age of onset and the rapidly progressive clinical course suggests the presence of certain compensatory mechanisms to overcome the absence of HO-1 function. The devastating clinical courses experienced by these patients indicate the critical importance of HO-1 in holding back rapidly progressive inflammation and organ dysfunction once an overwhelming inflammatory response is ignited by certain triggers. In all 5 Indian cases, laboratory findings were surprisingly similar, characterized by markedly increased platelet counts, leukocytosis and significant anemia. Levels of hepatic enzymes such as LDH, AST, and ALT uniformly showed significant elevations. Low to normal serum bilirubin and high haptoglobin concentration in the presence of hemolysis seem to be hallmarks of the illness. In addition to constantly elevated C-reactive protein (CRP) values, significant levels of serum ferritin were another characteristic of these cases. The cases also shared many clinical characteristics. However, clinical profiles were more variable than laboratory findings. Although asplenia and a prominent forehead were observed in all 5 cases, growth delay was not seen in Case 2 and Case 6. Hypertension was not seen in Case 6. Cerebral bleeding was observed only in Case 2 and Case 3. Arthralgia was seen in the first case, but was not detected in any of the 5 Indian cases.

2.3. The Iranian Case: A Novel HMOX1 Mutation Tahghighi et al. reported a 7th case of HO-1 deficiency, in a girl who was 17 months old when she presented with high fever, tachypnea, and respiratory distress [22]. She was the third child born to consanguineous Iranian parents. The first child in the proband’s family was a girl who was found to be completely healthy, but their second child was aborted spontaneously at 20 weeks for unknown reasons. On first admission, the patient Int. J. Mol. Sci. 2021, 22, 1514 6 of 16

revealed massive pericardial effusion without any evidence of infectious diseases or malignancies. Hepatomegaly was found and a liver biopsy showed iron deposits within the hepatic parenchyma. Laboratory findings included leukocytosis, increased platelet count, hemolytic anemia, and elevated levels of inflammatory markers. Increased levels of hepatic enzymes, triglycerides and ferritin were also noted. In addition to infectious diseases and malignancies, autoinflammatory diseases or immunodeficiencies were ruled out based on the clinical and laboratory findings. Corticosteroid proved ineffective for controlling inflammation and her condition deteriorated progressively. She died of recurrent fever, bleeding, heart failure, and ascites after 4 admissions. Due to the clinical profile and characteristics of laboratory data, Tahghighi et al. suspected hemophagocytic lymphohistiocytosis (HLH) or HO-1 deficiency. Post-mortem whole-exome sequencing revealed homozygous mutations of the HMOX1 gene (within exon 3, p.K204X). Both parents carried identical mutations on a single allele of the HMOX1 gene. Although the patient had a normal sized-spleen, other clinical and laboratory features were similar to those found in the Japanese (Case 1) and the 5 Indian cases (Cases 2–6). The characteristic laboratory features included leukocytosis, increased platelet count, anemia, elevated CRP, extremely high LDH and ferritin concentrations, hyperlipidemia and low to normal bilirubin levels. Clinical findings included prolonged or recurrent fever, hepatomegaly and hemolytic anemia. Although Tahghighi et al. did not perform any functional studies to confirm that the specific mutation resulted in defective enzyme function, ENTPRISE-X (http://cssb2.biology.gatech.edu/entprise-x/) predicted that mutations at codon 204 of HO-1 would be deleterious. A p.K204X mutation produces a truncated protein of 203 amino acids (instead of 288 amino acids) that is predicted to be highly pathological. The reason the patient with K204X mutation had a normal-sized spleen remains unclear. Tahghighi et al. did not mention if postmortem pathological examinations were performed to identify any structural abnormalities within the patient’s spleen. If HO-1 deficiency has any effect on splenic development, as suggested by the previous human cases and the findings from HO-1-knockout mice, the spleen would be highly expected to show significant pathological findings reflecting impaired splenic vascularity.

2.4. The Turkish Case: A Missense Mutation The case reported by Greil et al. (Case 8) represented the first case of missense mutation in the HMOX1 gene (p.G139V) [23]. The patient was the son of consanguineous Turkish parents. Clinical profiles were characteristic, in that he presented with microcytic anemia and progressive hepatosplenomegaly at 3 months old. The anemia proved resistant to iron supplementation and the patient was dependent on red cell transfusion. Liver biopsy revealed severe hemophagocytosis and Kupffer cell siderosis with extramedullary hematopoiesis. Slight hemophagocytosis was also seen in the bone marrow. Because of these clinical findings and the laboratory data, HLH was suspected and the patient received treatment with immunochemotherapeutic agents (HLH2004 protocol) with sustained remission of the signs of HLH [28]. However, elevated levels of inflammatory markers, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, ferritin, and CRP remained, indicating the sustained presence of an intense inflammatory reaction. DNA sequence analysis was performed to identify any known gene mutations re- sponsible for familial HLH, but failed to find any. Persistently elevated LDH despite low bilirubin levels led Greil et al. to analyze the HMOX1 gene, revealing the homozygous G139V mutation. This mutation was a missense mutation and expression of constitutive HO-1 was increased by cells, but HO-1 levels were not enhanced with additional oxidative stress. Apoptosis of peripheral blood mononuclear cells was examined upon exposure to oxidative stress (heme) and a significant difference in the level of cell death was seen between control and the patient’s cells, suggesting a functional defect of HO-1 activity in the patient’s cells. Although many features were similar to previously described cases of HO-1-deficient patients, some findings were distinct and not observed in other patients. Whereas HO-1 Int. J. Mol. Sci. 2021, 22, 1514 7 of 16

enzyme activity was significantly decreased, the mutation was associated with a gain of abnormal peroxidase function of the enzyme, resulting in increased urinary excretion of peroxidation products. Furthermore, mononuclear cells from the patient produced significantly high concentrations of inflammatory cytokines, such as TNF-α and IL-6, upon stimulation with lipopolysaccharide (LPS). These unique functional characteristics of mu- tant HO-1 resulted in distinct clinical features of this particular patient compared to other reported cases of human HO-1 deficiency with null-type mutations. Of particular interest is the fact that the patient with G139V mutation developed paradoxical inflammatory response to red cell transfusion, presumably due to abnormal peroxidase function provoked by constitutively enhanced expression of the mutated HO-1 product.

2.5. The Case from USA: A Compound Heterozygote of Paternal Frameshift and Maternal Splice Donor The most recent report of HO-1 deficiency by Chau et al. (Case 9) was characterized by interstitial lung disease associated with frequent flares of systemic inflammation [24]. The patient presented with the first inflammatory episode at 4 years of age with a one-month history of fatigue, intermittent fevers, and dark urine. Mild prognathism and slight frontal prominence were noted. High fever and hypoxemia persisted during hospitalization. The laboratory examination revealed leukocytosis, thrombocytosis, elevated LDH at 19,706 U/L, and hyperferritinemia to 1980 ng/mL. Hemolytic anemia with schistocytes and Howell- Jolly bodies were noted on peripheral blood smears. There was no evidence of autoimmune hemolytic anemia. Serum bilirubin level was normal. Abdominal CT scan revealed poorly perfused, hypoplastic spleen. Hepatomegaly was another characteristic finding and the liver biopsy revealed mild sinusoidal fibrosis, mild microvesicular steatosis, and increased iron deposit within Kupffer cells. His growth had been normal by 3 years of age. However, his growth slowed down significantly after the onset of the inflammatory episodes, being less than the 10th percentile for weight and 20th percentile for height at 8 years. Because of persistent and progressive respiratory symptoms, a biopsy of the lung was performed at 10 years and it demonstrated extensive fibrotic nonspecific interstitial pneumonia, patchy pleural fibrosis, and scattered cholesterol granulomas. Flares of inflammatory episodes were associated with fevers, respiratory symptoms and laboratory evidence of hemophagocytosis, suggesting macrophage activation syndrome or HLH. Because of these clinical characteristics, genetic testing for periodic fever syndromes and familial HLH was performed. However, no pathogenic mutation was identified in known genes. Therapy with corticosteroid, anti-IL-1R, anti-IL-6, and cyclosporine had been tried but with minimal benefit. He died at 10 years of age due to respiratory failure. Post-mortem whole exome sequencing was performed on the patient and the family members. It revealed that the patient had a compound heterozygous mutations of HMOX1, with paternal frame shift c.264_269delCTGG (p.L89Sfs*24) and maternal splice donor c.636 + 2T > A, consistent with HO-1 deficiency. The patient peripheral blood mononuclear cells did not produce HO-1 upon stimulation with cobalt protoporphyrin. Compared with the rest of the HO-1 deficiency cases, Case 9 is characterized by chronic and progressive lung disease. The pathological finding of the lung, pulmonary interstitial and intra-alveolar cholesterol granulomas, is a very rare finding. Chau et al. regard this as the sequela of pulmonary manifestation of small vessel vasculitis and macrophage activation. This case shows further phenotype expansion for HO-1 deficiency and at the same time, indicate the critical role of HO-1 in preventing inflammation and macrophage activation in various tissues and organs.

2.6. Denominators of Human HO-1 Deficiency Since Yachie et al. reported the first HO-1 deficiency patient, eight additional cases of HO-1 deficiency with nonsense or missense HMOX1 gene mutations have been described. A comparison of clinical characteristics is shown in Table1 and the laboratory features of these cases are shown in Table2. Why so few patients have been reported so far remains Int. J. Mol. Sci. 2021, 22, 1514 8 of 16

unclear. HO-1 may play cardinal roles during fetal growth and development, and most individuals with HO-1 deficiency may thus develop significant organ dysfunctions and die in utero or shortly after birth without being diagnosed. Unless the distinct clinical and laboratory features are recognized in time, thinking of HO-1 deficiency as a candidate diagnosis for the patient is extremely difficult, especially when the condition is still largely unfamiliar to clinicians. For this reason, proposing denominators of human HO-1 deficiency and recognizing this rare and difficult-to-treat disease early during the course of illness is of paramount importance.

Table 1. Clinical proﬁles of human HO-1 deﬁciency with null mutations of HMOX1.

Findings Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Sex male female male male female male female male male Age of 2 yr 15 yr 6 m 10 yr 7 yr 13 m 17 m 3 m 4 yr onset Age at 5 yr 16 yr 2 yr 10 yr 9 yr 20 m 3 yr 20 m 10 yr diagnosis Fever + + + + + + + + + Arthralgia + ------+ Hemolytic +++ ++ ++ ++ ++ ++ ++ ++ ++ anemia Jaundice ------Hematuria/ +/+ +/+ +/+ +/+ +/+ +/+ ? ? +/+ proteinuria Asplenia + + + + + + - splenomegaly hyposplenia Prominent + + + + + + ? ? + forehead Hypertension + + + + + - - - - Cerebral + + + ------bleeding Growth + - + + + + + ? + delay +: present, -: absent, +++: strongly positive, ++: moderately positive, ?: unknown or not described.

Table 2. Laboratory data of human HO-1 deﬁciency with null mutations of HMOX1.

Findings Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Mutations of exon 2 del R44X R44X R44X R44X R44X K204X G139V c.636+2T>A HMOX1 2-bp del (exon 3) (homo) (homo) (homo) (homo) (homo) (homo) (homo) p.L89Sfs24 Mother exon 2 del not done not done not done R44X/W * R44X/W K204X/W G139V/W c.636+2T>A/W Father exon 3; 2-bp del not done not done not done R44X/W R44X/W K204X/W G139V/W p.L89Sfs24/W CRP 6.7 30.8 5.3 normal 24.0 4.8 12.4 24.0 not shown (mg/dL) FDP-DD 186.1 >8 not done normal not done not done not done not done >20 (mg/mL) WBC (×103/mL) 51.6 18.5 38.0 39.6 not shown 43.2 33.0 19.9 53.8 Plt (×104/mL) 226 137 109 117 100 123 100 47.8 91.4 Ferritin (ng/mL) 780 4912 15,530 15,358 2500 >2000 27,425 4855 1980 LDH (IU/L) 17,470 9462 12,858 16,000 4000 21,400 15,350 15,713 19,706 AST/ 448/ 982/ 1080/ 689/ 300/ 652/ 580/ 301/ not shown ALT (IU/L) 74 149 283 68 80 133 813 74 Bilirubin 0.2 0.64 1.2 0.3 0.4 0.02 0.8 0.2–1.6 0.2 (mg/dL) * W; wild type.

Although ages at onset among the 9 HO-1 deficiency patients were quite variable, ranging from infancy to 15 years of age, laboratory data and clinical profiles were surprisingly uniform. Fever and hemolytic anemia were constant findings. Hematuria and proteinuria were observed in all of the Japanese and Indian cases. The reports on Cases 7 and 8 failed to mention whether the patients showed abnormal urine findings. The presence of hematuria or proteinuria would support the possibility of kidney injury in HO-1 deficiency. More importantly, bilirubin remained low or within normal ranges in all cases, despite the presence of active hemolytic anemia. Paradoxical normo- or hypobiliru- binemia in the presence of active hemolytic anemia is certainly the first denominator of HO-1 deficiency. Int. J. Mol. Sci. 2021, 22, 1514 9 of 16

Another distinct laboratory feature is the extremely elevated serum ferritin and LDH values. Although Cases 7, 8, and 9 showed at least transient episodes of HLH during the course of the illness, which may partly explain the high ferritin and LDH values, the clinical features were incompatible with typical HLH cases. More importantly, increases in leukocyte and platelet counts were invariably noted in all cases, which is contrary to the phenomenon observed in HLH patients. Extremely high ferritin and LDH values in the absence of apparent features of HLH may thus be the second denominator of HO-1 deficiency. Of particular importance, absence or hypoplasia of the spleen seems to be the hallmark of the illness, at least for the first 6 cases. Absence of the spleen seemed to be a specific finding for the first 6 HO-1-deficient cases and Case 9, but was not the case in Cases 7 and 8. Why these two cases showed a normal-sized spleen or splenomegaly, respectively, remains unclear. In this regard, a report by Kovtunovych et al. showed HO-1 mutation in mice resulting in progressive atrophy of the spleen due to fibrotic changes in the splenic artery with age, providing food for thought [29]. Although not described in the reports, knowing whether Cases 7 and 8 showed any evidence of splenic functional deficit would be of interest. Asplenia is certainly the most important denominator of human HO-1 deficiency if recognized in the absence of congenital heart disease. However, the possibility of HO-1 deficiency should not be excluded because the patients showed a normal-sized or enlarged spleen. If cases are accumulated in the future, a characteristic facial appearance may be able to be defined, but at present it is difficult to regard a prominent forehead or eyelid edema as a uniform characteristic of HO-1 deficiency patients. Hypertension and intracranial hemorrhage are certainly related to the basic pathology of HO-1 deficiency. However, these symptoms were not constantly observed in HO-1 deficiency patients. Information must be collected from a large number of cases to extend the list of denominators for the correct diagnosis of patients.

3. Animal Models of HO-1 Deficiency Poss and Tonegawa reported on HO-1-knockout mice with a C57BL/6 background in 1997, showing that these animals lacked the ability to reuse iron and were characterized by progressive anemia, tissue iron deposition, chronic inflammation, and delayed growth [25,26]. Serum iron levels were abnormally low. In contrast, there was intense accumulation of iron within the renal tubular epithelium and hepatic parenchyma, con- tributing to macromolecular oxidative damage, tissue injury, and chronic inflammation. Progressive increases in serum ferritin were seen along with progression of anemia and leukocytosis. Although spleen size was normal at birth, marked enlargement of the spleen developed as mice aged. Furthermore, these mice were extremely sensitive to oxidative injury and were prone to death with LPS administration. Embryonic fibroblasts derived from HO-1-knockout mice showed increased sensitivity to oxidative stress, such as hemin, H2O2, paraquat, and heavy metals. All these results indicated that HO-1 is important for iron homeostasis and indispensable for the rapid protection of cells and tissues from potential oxidative damage during stress. Kovtunovych et al. published a detailed study on the iron distribution and pathology of another strain of HO-1-knockout mice using a mixed C57BL/6-FVB background [29]. They showed progressive death of macrophages in the liver and spleen throughout the process of erythrophagocytosis and the resultant heme release in vivo, causing significant damage to the organs and intense inflammation of the surrounding tissues. In contrast to the splenomegaly seen in HO-1-knockout mice reported by Poss and Tonegawa, the spleen in these mice showed initial splenomegaly when young, but progressive atrophy as the mice aged. Pathological examination revealed initial enlargement of the red pulp of the spleen underwent progressive fibrotic changes and eventual atrophy and functional hyposplenism in older mice. Impaired splenic function was confirmed by the appearance of Howell-Jolly bodies within erythrocytes. As seen in human HO-1 deficiency patients and in the first Int. J. Mol. Sci. 2021, 22, 1514 10 of 16

strain of HO-1-knockout mice, iron deposition was prominent within the renal proximal tubules and hepatic parenchymal cells. Reduction of the Hb-Hp scavenger receptor, CD163, by macrophages was also noted, further indicating dysfunction of macrophages as the important producers of HO-1. A rat model of HO-1 deficiency was recently described by Atsaves et al. [30]. Sprague- Dawley rats were employed in the study to reveal the role of HO-1 in various forms of kidney disease. This rat model showed characteristic renal and extrarenal phenotypes. These rats showed hemolytic anemia with morphological abnormality of the circulating erythrocytes, such as poikilocytosis and the presence of target cells and acanthocytes. Splenomegaly, leukocytosis and impaired growth were also observed. Most rats died early, by 6 months old. Whether the observed splenomegaly was transient or would result in progressive atrophy (as shown in the mouse model of Kovtunovych et al. [29]) was not clear from their data, because the study did not extend beyond 24 weeks old. Renal dysfunction with structural abnormality and proteinuria was another characteristic finding. Glomerular damage was characterized by increased mesangial matrix and lesions of focal and segmen- tal sclerosis. Electron microscopy of the glomeruli revealed edematous podocytes with scant areas of foot process effacement. In contrast to human cases or mice models, iron deposition was not increased within the tubular epithelium or hepatic parenchyma of the rat model. In contrast to the progressive iron deposition in macrophages within the splenic red pulp, little iron deposition was observed even in older HO-1-deficient rats. Table3 summarizes the characteristics of HO-1-targeted mice in comparison with 8 human cases of HO-1 deficiency. In addition to intense inflammation and hemolytic anemia, abnormal iron deposition within renal tubular cells, and hepatic parenchymal cells, elevated levels of ferritin were also commonly observed in both mouse models and human cases. Although the spleen was detectable or enlarged in two of the human cases and in mouse models, asplenia or abnormal splenic pathology may also be a hallmark of HO-1 deficiency.

Table 3. Mouse models and human cases of HO-1 deﬁciency.

Mouse Models Human Cases Findings Poss et al. Kovtunovych Japanese Indian Cases Iranian Case Turkish Case USA Case [25,26] et al. [27] Case exon 2 del Gene R44X K204X G139V c.636 + 2T>A knockout knockout 2-bp del mutations (homo) (homo) (homo) p.L89Sfs24 (exon 3) Type of large large nonsense nonsense nonsense missense nonsense mutations deletion deletion mutation mutation mutation mutation mutation Anemia + + + + + + + RBC anomaly ? + + + ? + + splenomegaly Spleen splenomegaly asplenia asplenia normal size splenomegaly hyposplenia → atrophy Inﬂammation ++ ++ ++ ++ ++ ++ ++ Iron deposit ++ ++ ++ ? ++ ? ++ Low bilirubin ? ? yes yes yes yes yes Ferritin high high high high high high high LDH ? ? high high high high high Platelet ? increased increased increased increased normal increased +: present, -: absent, ++: moderately positive, ?: unknown or not described.

Based on reports describing human cases of HO-1 deficiency and animal models of HO-1 gene knockout, the spleen seems to show two potential fates in the absence of functional HO-1 protein. One extreme is the loss of splenic tissue early in utero. In these cases, the spleen is undetectable at birth, as seen in the first 6 cases of human HO-1 deficiency. The other extreme is progressive splenomegaly after birth and later atrophy of the spleen secondary to fibrosis of the red pulp (Figure4). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 17

Table 3. Mouse models and human cases of HO-1 deficiency.

Mouse Models Human Cases Findings Poss et al. Kovtunovych et Indian Iranian Japanese Case Turkish Case USA Case [25,26] al. [27] Cases Case exon 2 del Gene muta- R44X K204X G139V c.636 + 2T>A knockout knockout 2-bp del (exon tions (homo) (homo) (homo) p.L89Sfs24 3) Type of muta- large large nonsense nonsense nonsense missense nonsense tions deletion deletion mutation mutation mutation mutation mutation Anemia + + + + + + + RBC anomaly ? + + + ? + + splenomeg- splenomegaly normal Spleen asplenia asplenia splenomegaly hyposplenia aly → atrophy size Inflammation ++ ++ ++ ++ ++ ++ ++ Iron deposit ++ ++ ++ ? ++ ? ++ Low bilirubin ? ? yes yes yes yes yes Ferritin high high high high high high high LDH ? ? high high high high high Platelet ? increased increased increased increased normal increased +:present, -:absent, ++:moderately positive, ?:unknown or not described.

Based on reports describing human cases of HO-1 deficiency and animal models of HO-1 gene knockout, the spleen seems to show two potential fates in the absence of functional HO-1 protein. One extreme is the loss of splenic tissue early in utero. In these cases, the spleen is undetectable at birth, as seen in the first 6 cases of human HO-1 deficiency. Int. J. Mol. Sci. 2021, 22, 1514 11 of 16 The other extreme is progressive splenomegaly after birth and later atrophy of the spleen secondary to fibrosis of the red pulp (Figure 4).

Figure 4. HO-1 deficiency and splenic development. In human cases of HO-1 deficiency with Figure 4. HO-1 deficiency and splenic development. In human cases of HO-1 deficiency with mu- mutations found in Japan and India, the patients were found to be asplenic, presumably reflecting tations found in Japan and India, the patients were found to be asplenic, presumably reflecting intrauterine defects in splenic development. In mouse models of HO-1 deficiency and human cases intrauterine defects in splenic development. In mouse models of HO-1 deficiency and human caseswith with mutations mutations found found in Turkey in Turkey and Iran,and Iran, the spleens the spleens showed showed transient transient splenomegaly splenomegaly followed fol- by lowedprogressive by progressive atrophy afteratrophy birth. after birth. 4. HO-1 Deficiency and Oxidative Stress

Pathological examination of the first case of HO-1 deficiency revealed characteristic tissue injury. Of particular interest was the fact that cell injury was not observed ubiqui- tously, but instead confined to selected organs or specific cell types. Among these, the kidneys, liver, circulating monocytes, and vascular endothelial cells are the tissues showing apparent injury and dysfunction [31]. In the first human case, mild mesangial proliferation and thickening of the capillary loop were observed within the glomeruli. Electron microscopy revealed marked swelling and detachment of endothelial cells throughout the glomerular capillary. In addition to the glomerular damage, tubulointerstitial injury with tubular atrophy was significant. The liver was massively enlarged and significant amyloid accumulation was evident, resulting in marked atrophy of hepatocytes. Scattered foci of iron deposits were observed in both the kidneys and liver. The cytoplasm of circulating monocytes was vacuolated and monocyte surface antigens were significantly different from normal profiles. This selective organ damage in HO-1 deficiency seems to have several causes. First, these susceptible cells are constantly exposed to oxidative stress, for both anatomical and functional reasons. Vascular endothelial cells are the recipients of shear stress and are exposed to multiple oxidative stressors, including hemolysis, pH changes, and hypoxemia. In the HO-1 deficiency patient, secondary accumulation of heme proteins, cholesterol and fragmented erythrocytes further aggravates these oxidative stresses. Renal tubular cells are constantly exposed to hematuria, proteinuria and various other substances excreted in urine. Resident macrophages and circulating monocytes are frequently activated to perform scavenger functions, and also function as central players in both innate and acquired immunity. Second, these cells may be particularly sensitive to oxidative injury, but at the same time, may also serve as a high-quality sensor of oxidative stress for the organs. Oxidative insults to these susceptible cells rapidly induce release of anti-oxidative molecules, saving the organs from critical damage. Last, these cells, because they have to serve as oxidative stress sensors, have some capacity for neutralization to combat these insults. HO-1 may play roles as a central molecule in all these mechanisms. Another distinct feature of the first case of human HO-1 deficiency was the defective endothelial function, as represented by extremely abnormal parameters for coagulation Int. J. Mol. Sci. 2021, 22, 1514 12 of 16

and fibrinolysis. Compared to other hematological illnesses associated with dissemi- nated intravascular coagulation, the patient exhibited extraordinarily elevated values for thrombin-antithrombin complex, fibrinogen degradation products, and palsmin-α2 plasmin inhibitor complex. Paradoxically, platelet numbers were constantly elevated. The data indicated that HO-1 or HO-1 products, such as CO, may be associated with regulation of the coagulation/fibrinolytic system. We recently demonstrated in in vitro cultures that a CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer suppressed TNF-α- induced up-regulation of tissue factor and plasminogen activator inhibitor type 1 by human umbilical vein endothelial cells. The CO-releasing molecule also suppressed activation of mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways by TNF-α. LPS-induced TNF-α production by circulating mononuclear cells was also significantly inhibited by the CO-releasing molecules [32]. These results may well explain the characteristic findings seen in HO-1 deficiency patients. At the same time, these findings support the view that CO-releasing molecules may provide a novel anti-coagulative and Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 17 anti-inflammatory therapy [33,34]. A summary of macrophage activation and endothelial cell dysfunction is shown in Figure5 . The lack of HO-1 resulted in unregulated activation of macrophages and sub- tionsequent of the coagulation/fibrinolysis release of excessive inflammatory system. Figure cytokines. 5 illustrates At the the same role time, of HO-1 HO-1 as deficiency an in- hibitorresulted of cytokine in overproduction overproduction of tissue and endo factorthelial by endothelial cell dysfunction cells, leadingassociated to abnormalwith cata- ac- strophictivation tissue of the injury coagulation/fibrinolysis seen in systemic inflammatory system. Figureresponse5 illustrates syndrome. the role of HO-1 as an inhibitor of cytokine overproduction and endothelial cell dysfunction associated with catastrophic tissue injury seen in systemic inflammatory response syndrome.

Figure 5. HO-1 deficiency and oxidative stress. Pathogen-associated molecular patterns (PAMPs) Figurebind 5. toll-like HO-1 deficiency receptors (TLRs)and oxidative on vascular stress. endothelial Pathogen-associated cells or monocytes. molecular Lack patterns of HO-1 (PAMPs) results in un- bindregulated toll-like activationreceptors (TLRs) of macrophages on vascular with endothe excesslial release cells of or inflammatory monocytes. Lack cytokines. of HO-1 Hemophagocytic results in unregulatedlymphohistiocytosis activation of (HLH) macrophages or macrophage with exce activationss release syndrome of inflammatory (MAS) may cytokines. result. At Hemopha- the same time, gocyticHO-1 lymphohistiocytosis deficiency results in (HLH) overproduction or macrophage of tissue activation factors bysyndrome endothelial (MAS) cells, may leading result. to At abnormal the sameactivation time, HO-1 of the deficiency coagulation/fibrinolysis results in overproducti system,on and of disseminatedtissue factors by intravascular endothelial coagulation cells, leading (DIC). to abnormalProlonged activation and sustained of the activation coagulation/fibrin of monocytes,olysis plateletssystem, and dissem endothelialinated cells intravascular leads to exhaustion coagulation (DIC). Prolonged and sustained activation of monocytes, platelets and endothelial cells and dysfunction of these cells. leads to exhaustion and dysfunction of these cells. 5. Novel Anti-Inflammatory Therapies 5. Novel Anti-Inflammatory Therapies Monocytes/macrophages comprise at least two functionally distinct subsets, M1 andMonocytes/macrophages M2 [35–37]. The different comprise subsets at of least monocyte/macrophage two functionally distinct lineage subsets, differentiate M1 and in M2response [35–37]. toThe environmental different subsets stimuli. of monocyte/macrophage M1 macrophages are lineage the “classical” differentiate macrophages, in re- sponserepresenting to environmental the pro-inflammatory stimuli. M1 macrop subset,hages whereas are M2the “classical” macrophages macrophages, are “alternatively” rep- resentingactivated the macrophages pro-inflammatory that resolve subset, inflammatory whereas M2 responses,macrophages perform are “alternatively” scavenger functions ac- tivatedand promotemacrophages tissue that remodeling resolve inflammato and repair. Interferon-ry responses,γ is perform the key cytokinescavenger driving functions the M1 andpathway, promote whereas tissue remodeling IL-4, IL-10, and repair. steroids Interferon- promote monocyteγ is the key differentiation cytokine driving into the the M2 M1lineage pathway, [38 ,whereas39]. IL-4, IL-10, and steroids promote monocyte differentiation into the M2 lineage [38,39]. We have previously reported that circulating monocytes produce significant levels of HO-1 during Kawasaki disease and infectious diseases, suggesting a certain anti-inflammatory role during inflammatory illnesses [40]. Furthermore, we investigated the profiles of cytokine mRNA expression in two subsets of circulating monocytes [41]. In that study, freshly isolated CD16high CCR2negative monocytes expressed significant levels of HO- 1 mRNA in vivo. They produced little IL-10 on stimulation with LPS. In contrast, a major subset of CD16low CCR2positive monocytes did not express HO-1 mRNA in vivo, but responded significantly to LPS and produced IL-10. The fractions of CD16high CCR2negative monocytes increased during various acute inflammatory diseases, such as Kawasaki disease and influenza virus infection, suggesting the anti-inflammatory roles played by monocytes through HO-1 production. In macrophages and dendritic cells, CO reduces proinflammatory and increases anti- inflammatory cytokine secretion in response to LPS [42,43]. HO-1-mediated anti-inflammatory effects may therefore be closely linked to anti-inflammatory mechanisms, such as the suppression of immune and inflammatory responses in macrophages via diminished antigen-presenting capacity and cytokine synthesis [44,45]. This is consistent with our

Int. J. Mol. Sci. 2021, 22, 1514 13 of 16

We have previously reported that circulating monocytes produce significant levels of HO-1 during Kawasaki disease and infectious diseases, suggesting a certain anti- inflammatory role during inflammatory illnesses [40]. Furthermore, we investigated the profiles of cytokine mRNA expression in two subsets of circulating monocytes [41]. In that study, freshly isolated CD16high CCR2negative monocytes expressed significant levels of HO-1 mRNA in vivo. They produced little IL-10 on stimulation with LPS. In contrast, a major subset of CD16low CCR2positive monocytes did not express HO-1 mRNA in vivo, but responded significantly to LPS and produced IL-10. The fractions of CD16high CCR2negative monocytes increased during various acute inflammatory diseases, such as Kawasaki disease and influenza virus infection, suggesting the anti-inflammatory roles played by monocytes through HO-1 production. In macrophages and dendritic cells, CO reduces proinflammatory and increases anti-inflammatory cytokine secretion in response to LPS [42,43]. HO-1-mediated anti- inflammatory effects may therefore be closely linked to anti-inflammatory mechanisms, such as the suppression of immune and inflammatory responses in macrophages via diminished antigen-presenting capacity and cytokine synthesis [44,45]. This is consistent with our finding in HO-1 deficiency patients, in whom the lack of HO-1 resulted in a marked rise in circulating heme and subsequent oxidative vascular and tissue injuries, anemia, and chronic inflammation. HO-1 is induced by CD163-mediated uptake of the Hb-Hp complex [46–48]. Schaer et al. reported that macrophages express upregulated levels of CD163 in sepsis-induced hemophagocytic syndrome [49]. These macrophages expressed significant levels of HO-1, suggesting their role as a negative regulator of inflammation. We demonstrated that serum HO-1 concentrations correlate closely with serum sCD163 concentrations, and these are extremely high in the macrophage activation syndrome associated with sJIA [50]. These findings indicate that serum HO-1 might be derived from CD163+ alternatively activated macrophages. In particular, hemophagocytic macrophages may represent a major source of HO-1 in sJIA. Increased levels of serum HO-1, as well as CD163, suggest that alternative activation of macrophages is switched on in sJIA. For many years, various anti-inflammatory agents, including steroids, non-steroidal anti-inflammatory drugs (NSAIDs), and immunosuppressants have been the main agents used to control excessive inflammation and tissue/organ injury. These agents are effective in some cases, but conventional anti-inflammatory/immunosuppressive therapies resulted in therapeutic failure in HO-1 deficiency cases for two reasons. One important and critical reason is that inflammation and immune reactions are, despite causing extensive tissue injury, organ damage, and discomfort to the patient, the critical components of host responses to pathogens and insults. Non-judicious use of conventional drugs may result in the host failing to expel pathogens and instead succumbing to overwhelming infection or uncontrollable cancer growth. Second, when the tissues and organs are already significantly damaged after the pathogen insult and subsequent host immune responses, addition of anti-inflammatory or immunosuppressive agents may further aggravate the host status. Understanding the built-in mechanism protecting cells and organs from oxidative injury, I would like to propose the third alternative of anti-inflammatory therapy to promote the anti-oxidative functions of each cell. The prime target of such therapy is HO-1. Pharmacological induction of cellular HO-1 production, use of CO-releasing molecules, or enhancement of Hb-Hp receptor expression by steroid are examples of these approaches. As Naito et al. recently proposed, several pharmacological agents can drive a phenotypic shift to M2 macrophages and enhanced HO-1 production in vivo [51]. In a recent review on haptoglobin, di Masi et al. stress the anti-inflammatory role of haptoglobin in various clinical settings. Hp not only regulates Hb clearance from circulation, but at the same time modulates the immune status of the host [52]. Considering the role of haptoglobin as the modulator of inflammation, it becomes a potential target of anti-inflammatory therapy. Int. J. Mol. Sci. 2021, 22, 1514 14 of 16

The disease caused by SARS-CoV-2 is becoming a common burden worldwide as the numbers of the patients increase progressively. Although the most patients recover without significant complication, small, but significant proportion of the patients with COVID-19 experience a broad array of organ damage. Accumulating evidence suggest that the major pathology of COVID-19 is characterized not only by acute respiratory distress syndrome, but at the same time excessive systemic inflammatory reaction and coagulopathy [53]. Considering these unique pathology of COVID-19, pharmacological induction of HO-1 in vivo may certainly serve as a potential therapeutic target to overcome the severe complication of COVID-19 [54–56].

6. Conclusions Collective data obtained from 9 human cases of HO-1 deficiency and HO-1-knockout animal models support the view obtained from in vitro experiments. HO-1 plays cardinal roles in abrogating oxidative stress and inflammation, thus ameliorating cell and tissue injuries. Pharmacological manipulation of HO-1 production may provide an effective intervention to regulate inflammation and protect organs without introducing immuno- suppression or cytocidal agents. Furthermore, recognition of denominators for HO-1 deficiency will help in identifying this extremely rare, underdiagnosed disease at an early stage. Avoidance of exogenous stress along with appropriate treatment may prevent early death in these patients.

Funding: This work was supported by JSPS KAKENHI Grant Number 15H04873. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Kanazawa University Graduate School of Medical Sciences. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Conﬂicts of Interest: The author has no conﬂict interests to declare.

Abbreviations

HO heme oxygenase CO carbon oxide Hb hemoglobin Hp haptoglobin

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